Vitamin B12 nasal spray and method of use

ABSTRACT

The present invention relates generally to vitamin B 12  nasal spray compositions and methods of using the same in the treatment of vitamin B 12  deficiency and various disorders that are related to such deficiency. In particular embodiments, the present invention is directed to treatment methods comprising intranasal administration of a cobalamin composition according to a particular dosing and frequency schedule and to a preservative-free nasal spray composition comprising a cobalamin compound useful in the practice of such treatment methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/709,200 filed Aug. 17, 2005, the entire content of which ishereby incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to vitamin B₁₂ nasal spraycompositions and methods of using the same in the treatment of vitaminB₁₂ deficiency and various disorders that are related to suchdeficiency. In particular embodiments, the present invention is directedto treatment methods comprising intranasal administration of a cobalamincomposition according to a particular dosing and frequency schedule andto a preservative-free nasal spray composition comprising a cobalamincompound useful in the practice of such treatment methods.

BACKGROUND

Vitamin B₁₂ is a water soluble vitamin that plays a role in mammaliangrowth, hematopoiesis, production of epithelial cells, and maintenanceof the nervous system. It was first isolated from liver concentrate in1948 and structurally elucidated in the late 1950's.

Cyanocobalamin is a form of vitamin B₁₂ and is one of the class of B₁₂vitamins or cobalamin compounds that includes vitamin B_(12a)(hydroxocobalamin), vitamin B_(12b) (aquacobalamin), vitamin B_(12c)(nitrilocobalamin), methyl B₁₂ (methylcobalamin) and coenzyme B₁₂(5′deoxyadenosine cobalamin). Cyanocobalamin and hydroxocobalamin arethe principal members of the class and the most widely employed incompositions used to treat vitamin B₁₂ deficiency and disorders that arerelated to this deficiency. Such disorders include anemias (mostcommonly pernicious anemia) and diphyllobothrium latum (fish tapeworm)infestation of the intestine, a disorder with symptomology that mimicspernicious anemia.

Several routes of administration of vitamin B₁₂ are known. Among theseare parenterally, including intramuscular and subcutaneous injection,orally as a component of a tablet or solution, and nasally, as acomponent of a nasal spray or gel. Although the minimum daily dietaryrequirement of vitamin B₁₂ is approximately 0.1 μg for a healthy human,therapeutic administration of vitamin B₁₂ is typically in significantlylarger doses. For example, the prescribed initial therapeutic dose isgenerally from about 100 μg to about 1000 μg, and is most oftenadministered by intramuscular injection. Subsequent vitamin B₁₂maintenance therapy may be by injection or by oral administration of acobalamin composition. Use of vitamin B₁₂ injections for maintenancetherapy has obvious disadvantages, including the inconvenience and painassociated with the injection that typically must be administered bymedical personnel. Orally administered cobalamin compositions may failto be adequately absorbed in the patient, particularly in those in whichsecretion or utilization of intrinsic factor is inadequate.

Intranasal administration of cobalamin compositions for vitamin B₁₂maintenance therapy offers advantages over these alternative routes ofadministration. Typically, such therapy includes relatively infrequent,high dose nasal administration of a cobalamin composition. For example,NASCOBAL nasal spray solution containing 0.5% by weight cyanocobalaminis administered in one nostril once weekly in a dose of 500 μg (i.e.,500 μg cyanocobalamin per 0.1 mL actuation of the spray bottle pump).

Although maintenance therapy with vitamin B₁₂ nasal compositions hasproven generally effective, those undergoing such therapy may experiencesome irritation of the nasal mucosa and discomfort due, in part, to thehigh dosage of the cobalamin compound typically administered as well aspreservatives and other additives often present in these compositions.Moreover, effective vitamin B₁₂ maintenance therapy would be enhanced ifmore stable or even blood serum levels of vitamin B₁₂ could be attainedthrough intranasal administration of a cobalamin composition.Accordingly, a need persists for improvements in vitamin B₁₂ nasalcompositions and methods of nasal administration of such compositions inthe treatment of vitamin B₁₂ deficiency and various vitamin B₁₂deficiency-mediated disorders.

SUMMARY OF THE INVENTION

Among the various aspects of the present invention is the provision of amethod of frequent, low dose nasal administration of a vitamin B₁₂composition and an improved vitamin B₁₂ composition suitable for use insuch methods of treatment.

Briefly, therefore, the present invention is directed to a method formaintaining vitamin B₁₂ blood serum levels in a mammals, the methodcomprising nasally administering to the mammal at least once every threedays an aqueous composition comprising a cobalamin compound in an amountsufficient to deliver a dose of no more than about 150 μg of thecobalamin compound to the mammal.

The present invention is further directed to a method for maintainingnormal hematologic status in a pernicious anemia patient followingintramuscular vitamin B₁₂ injection therapy, the method comprisingnasally administering to said patient at least once every three days anaqueous composition comprising a cobalamin compound in an amountsufficient to deliver a dose of no more than about 150 μg of thecobalamin compound to the patient.

The present invention is further directed to a composition for nasaladministration of a cobalamin compound, the composition comprising anaqueous solution containing a cobalamin compound and a pharmaceuticallyacceptable buffer, the composition having a pH of at least about 6.5.

The present invention is further directed to a composition for nasaladministration of a cobalamin compound, the composition comprising anaqueous solution containing a cobalamin compound and a pharmaceuticallyacceptable buffer and being free of preservatives.

Other objects and features will be in part apparent and in part pointedout hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the mean serum vitamin B₁₂ levels and 95% confidenceinterval for all six visits in the study conducted in the Example.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part on the observation thatconventional intranasal administration of cobalamin compositions maylead to irritation of the nasal membranes or mucosa. Irritation may becaused or exacerbated by the relatively high dose of cobalamin compoundtypically administered and/or preservatives and other additives commonlycontained in these compositions. This irritation, compounded byinfrequent, high dose administration of conventional intranasalcobalamin compositions (e.g., anywhere from once a week to as rarely asonce a month), can lead to patient noncompliance and variable bloodserum vitamin B₁₂ levels and ultimately to inconsistent or ineffectivetreatment of vitamin B₁₂ deficiency-mediated disorders.

In order to provide patients with a more consistent and effectivetreatment for vitamin B₁₂ deficiency and related disorders, the presentinvention is directed to methods for maintaining vitamin B₁₂ blood serumlevels in a mammal comprising more frequent intranasal administration ofa cobalamin composition and at lower dosages than previously recognizedas effective in the treatment of such disorders. The present inventionfurther provides compositions containing a cobalamin compound useful inthe practice of the treatment methods disclosed herein, while minimizingthe risk of irritation of the nasal mucosa and patient noncompliancewith the prescribed treatment regimen.

The treatment methods disclosed herein are generally applicable formaintenance of vitamin B₁₂ blood serum levels in a patient in need ofvitamin B₁₂ therapy. Such a patient may be suffering from a vitamin B₁₂deficiency, a disorder resulting from such a deficiency, or a disordermimicking the symptomology of such a deficiency. Examples of disordersresulting from or mimicking a vitamin B₁₂ deficiency include, forexample, anemia, including pernicious anemia; nerve degeneration,typically as a result of degradation or lack of myelin; and infestationby intestinal parasites or bacteria such as diphyllobothrium latum (fishtapeworm) that absorb large quantities of vitamin B₁₂ in the host. Otherindications for application of the treatment methods disclosed hereininclude, for example, maintenance of normal hematologic status inpernicious anemia patients in remission subsequent to intramuscularvitamin B₁₂ injection therapy and who have no nervous systeminvolvement; remedying vitamin B₁₂ dietary deficiencies (e.g., invegetarians); treatment of patients suffering from vitamin B₁₂malabsorption phenomena such as that resulting from inadequate secretionand/or utilization of intrinsic factor (e.g., due to HIV infection,AIDS, Crohn's disease, tropical and nontropical sprue, extensiveneoplasia, subtotal or total gastrectomy, etc.); maintenance of vitaminB₁₂ in excess of normal dietary requirements due to pregnancy, renaldisease, thyrotoxicosis, hemolytic anemia, hemorrhage, etc; and patientshaving elevated serum homocysteine, cystathionine, methylmalonic acidand/or 2-methylcitric acid levels.

The present invention is directed particularly to the treatment ofhumans in need of vitamin B₁₂ therapy. However, it should be understoodthat the methods disclosed herein are generally applicable to thetreatment of mammals including, for example, domesticated house pets,such as dogs and cats, as well as farm animals, such as cattle, pigs,horses, sheep and goats.

Methods for Intransal Administration of a Cobalamin ContainingComposition

Generally, the treatment methods of the present invention comprise morefrequent and lower dose intranasal administration of a compositioncontaining a cobalamin compound. It has been discovered that morefrequent and lower dose intranasal administration of a cobalamincomposition offers several advantages over conventional intranasalvitamin B₁₂ maintenance therapy, including reduced risk of irritation ofthe nasal mucosa and more stable or even vitamin B₁₂ blood serum levels(i.e., smaller peak to trough variances) by more closely mimicking thetypical mammalian dietary intake of vitamin B₁₂. Moreover, it isbelieved that patient compliance is improved by treatment regimensincluding more frequent administration of the cobalamin composition. Asdescribed in greater detail below, the compositions used in the practiceof the methods disclosed herein include, for example, aqueous solutionsof the cobalamin compound along with various optional components such asisotonicity agents, buffering agents, humectants, surfactants andpreservatives.

The treatment method for maintaining vitamin B₁₂ blood serum levels in amammal in accordance with the present invention comprises nasallyadministering to the mammal an aqueous composition comprising acobalamin compound at least once every three days, preferably at leastonce every two days. The frequency of administration may vary during thetreatment period. For example, the composition may be administered everyday during an initial portion (e.g., the first week or first two weeks)of the treatment period (e.g., daily during an initial “loading” periodto increase vitamin B₁₂ blood serum levels), and then at less frequentintervals during the remainder of the treatment period. However, inorder to promote patient compliance, administration preferably occurs atregular intervals throughout the treatment period. In accordance with amore preferred embodiment, the treatment method of the present inventioncomprises daily intranasal administration (i.e., one or moreadministrations per day), and especially once daily, intranasaladministration of the cobalamin composition during the treatment period.

The dose of the cobalamin compound delivered to the patient with eachadministration will vary generally with the frequency of administration(a higher dose being utilized with longer intervals betweenadministrations), as well as with the needs of the patient and the typeof disorder being treated, as would be apparent to those skilled in theart, but is generally lower than the dosing conventionally employed.Generally, the dose of the cobalamin compound delivered to the patientwith each administration is no more than about 150 μg, preferably nomore than about 125 μg, more preferably no more than about 100 μg, andstill more preferably no more than about 75 μg of the cobalamincompound, and is at least 5 μg, preferably at least about 10 μg of thecobalamin compound.

With respect to a preferred treatment regimen comprising dailyintranasal administration of a cobalamin composition, the daily dose ofthe cobalamin compound delivered to the patient is preferably from about5 μg to about 100 μg, more preferably from about 20 μg to about 80 μg,still more preferably from about 30 μg to about 70 μg and even morepreferably from about 40 μg to about 60 μg. In accordance with anespecially preferred embodiment comprising once daily intranasaladministration of the cobalamin composition, the dose of the cobalamincompound delivered to the patient with each administration is usually nomore than about 80 μg. In such an embodiment, the dose of the cobalamincompound delivered to the patient with each administration is preferablyfrom about 5 μg to about 80 μg, more preferably from about 10 μg toabout 60 μg, more preferably from about 15 μg to about 50 μg, morepreferably from about 20 μg to about 50 μg, more preferably from about30 μg to about 50 μg, still more preferably from about 40 μg to about 50μg and even more preferably from about 45 μg to about 50 μg. Forexample, in an embodiment comprising daily intranasal administration ofthe cobalamin composition, the dose of the cobalamin compound deliveredto the patient per administration may be about 10 μg, about 15 μg, about20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg,or about 50 μg.

In accordance with another embodiment of the treatment method disclosedherein comprising once daily intranasal administration of the cobalamincomposition, the dose of the cobalamin compound delivered to the patientwith each administration is from about 5 μg to about 45 μg, preferablyfrom about 10 μg to about 45 μg, more preferably from about 15 μg toabout 45 μg, more preferably from about 20 μg to about 45 μg, still morepreferably from about 30 μg to about 45 μg, and even more preferablyfrom about 40 μg to about 45 μg.

With respect to the dose of the cobalamin compound delivered to thepatient per administration, it should be understood that eachadministration may comprise one or a plurality of applications or spraysof the cobalamin composition delivered to the nasal mucosa of thepatient through one or both nostrils, the number of applications orsprays being dependent upon the concentration of the cobalamin compoundin the composition, the quantity of the composition delivered per spray,and the desired dose per administration as readily determined by oneskilled in the art. As described in greater detail below, the cobalamincomposition is preferably dispensed from a spray bottle including a pump(e.g., a manually actuated pump) capable of delivering a metered sprayof the cobalamin composition of predetermined volume (typically about0.1 mL). Furthermore, and by way of example, a daily dose of 50 μg of acobalamin compound may be administered in a single administrationcomprising one or more applications or metered sprays containing a totalof 50 μg of cobalamin compound (e.g., a single administration comprisingtwo applications or metered sprays, one in each nostril and eachcontaining 25 μg of cobalamin compound) or in multiple administrations(e.g., four administrations at six hour intervals, each administrationcomprising one or more applications or metered sprays, in one or bothnostrils, each administration containing a total of 12.5 μg of cobalamincompound).

The weekly dose of the cobalamin compound received by the patient willgenerally not be in excess of about 1000 μg, more preferably about 800μg, still more preferably about 600 μg, even more preferably about 500μg, still more preferably about 450 μg, even more preferably about 400μg, and most preferably not in excess of about 350 μg of the cobalamincompound. Accordingly, in one embodiment, the patient may receive aweekly dosage of from about 50 μg to about 500 μg of the cobalamincompound. In another embodiment, the patient may receive a weekly dosageof from about 100 μg to about 450 μg of the cobalamin compound. In yetanother embodiment, the patient may receive a weekly dosage of fromabout 150 μg to about 400 μg of the cobalamin compound. In still anotherembodiment, the patient may receive a weekly dosage of from about 200 μgto about 400 μg of the cobalamin compound. In yet another embodiment,the patient may receive a weekly dosage of from about 250 μg to about350 μg of the cobalamin compound. In still another embodiment, thepatient may receive a weekly dosage of from about 300 μg to about 350 μgof the cobalamin compound. In a particularly preferred embodiment, thepatient may receive a weekly dosage of about 350 μg of the cobalamincompound, delivered by once daily nasal administration of 50 μg of acobalamin compound.

Compositions Containing A Cobalamin Compound

A further aspect of the present invention is a composition comprising acobalamin compound that may be used in a regimen to treat vitamin B₁₂deficiency and deficiency related disorders. The composition may be usedin previously known regimens of treatment for vitamin B₁₂ deficiency,but preferably is used in the practice of the treatment regimen of thepresent invention described above.

A composition of the present invention generally comprises an aqueoussolution containing a cobalamin compound. The aqueous solution ispreferably isotonic. The cobalamin may be any of a number of knowncobalamin compounds, including for example, cyanocobalamin,hydroxocobalamin (vitamin B_(12a)), hydroxocobalamin HCl, sulfate,acetate and other hydroxocobalamin salts, aquacobalamin (vitaminB_(12b)), nitrilocobalamin (vitamin B_(12c)), methylcobalamin (methylB₁₂), 5′-deoxyadenosine cobalamin (coenzyme B₁₂), pharmaceuticallyacceptable salts thereof, chemically modified equivalents thereof, andmixtures thereof. The selection of the specific form of cobalamin to beused in the composition depends upon a number of factors known to thoseof skill in the art, including, for example, the composition in whichthe cobalamin compound is to be mixed or dissolved, the amount orconcentration of cobalamin compound desired in the composition, thesolubility of the cobalamin compound and the pH of the composition. Inone embodiment, the cobalamin compound is cyanocobalamin USP. In anotherembodiment, the cobalamin compound is hydroxocobalamin. Theconcentration of the cobalamin compound in the composition can varydepending upon a number of factors known to those skilled in the art,including, for example, the composition in which the cobalamin compoundis to be mixed or dissolved, the solubility of the cobalamin compound,the pH of the composition, the means by which the cobalamin compositionis delivered to the nasal mucosa (e.g., using compressed gas or apropellant), the volume of the spray dispensed per application and thedesired dosage to be delivered to the patient. Generally, the cobalamincompound may be present in the composition in a concentration of no morethan about 20 weight percent (% w/w), for example, from about 0.0001%w/w to about 20% w/w, preferably from about 0.01% w/w to about 10% w/w,more preferably from about 0.02% w/w to about 1% w/w, still morepreferably from about 0.02% w/w to about 0.04% w/w and most preferablyabout 0.025% w/w.

The isotonicity of the composition may generally be achieved andmaintained using sodium chloride or another pharmaceutically acceptableisotonicity agent, such as, for example, dextrose, boric acid, sodiumtartrate, other organic or inorganic solutes and mixtures thereof. Theisotonicity agent is typically present in the composition in aconcentration sufficient to cause the osmolarity of the composition tobe from about 280 mOsmols to about 290 mOsmols±50 mOsmols. Sodiumchloride is generally preferred, particularly if a buffer containingsodium ions is used in the composition, and is typically present in anamount that is physiologically equivalent to the tonicity of the nasalmembranes.

The cobalamin composition of the present invention may further include apharmaceutically acceptable buffer in order to maintain the desired pH.Non-limiting examples of suitable buffers used to adjust and maintainthe pH of the composition include acetate, citrate, prolamine,phosphate, carbonate, phthalate, borate, or other pharmaceuticallyacceptable buffers and mixtures thereof. In a particular embodiment, thebuffer comprises sodium phosphate. The pH of the composition ismaintained generally to be compatible with the fluids of the nasalmembrane in order to minimize irritation. For example, the compositionmay be maintained at a pH from about 3 to about 11. In one embodiment,the composition may be maintained at a pH from about 3 to about 6.5,preferably from about 4 to about 6.5, more preferably from about 5 toabout 6.5, still more preferably from about 6 to about 6.5, and mostpreferably about 6.5. Alternatively, the composition may be maintainedat a pH greater than 6.5, preferably from about 6.5 to about 11, morepreferably from about 7 to about 10, still more preferably from about 7to about 9, even more preferably from about 7 to about 7.7, even morepreferably about 7.2, and most preferably about 7.7. The concentrationof the buffer in the composition will depend upon the selection of thebuffer and the desired pH.

The present composition may also contain various pharmaceuticallyacceptable additives such as tolerance enhancers (sometimes morespecifically referred to as humectants), absorption enhancers (sometimesalso referred to as surfactants), preservatives, viscosity modifyingagents (e.g., thickening agents), osmolarity adjusters, complexingagents, stabilizers, solubilizers, or any combination thereof.

A tolerance enhancer may be used in order to inhibit drying of the nasalmembrane or mucosa. A tolerance enhancer may also serve the purpose ofinhibiting or relieving irritation of the nasal membranes. Examples ofsuitable tolerance enhancers include, for example, humectants such assorbitol, propylene glycol, glycerol, glycerin, hyaluronan, aloe,mineral oil, vegetable oil, soothing agents, membrane conditioners,sweeteners, and mixtures thereof. The selection and concentration of atolerance enhancer may depend on a number of factors, including, forexample, the type and concentration of cobalamin compound being used inthe composition. When used, the concentration of the tolerance enhancerin the composition will typically be in amounts from about 0.01% w/w toabout 20% w/w.

A surfactant or absorption enhancer may also be used in the compositionin order to enhance the absorption of the cobalamin compound across thenasal membrane. Suitable absorption enhancers include non-ionic, anionicand cationic surfactants. Any of a number of well-known surfactants maybe used, including, for example, polyoxyethylene derivatives of fattyacids, partial esters of sorbitol anhydrides, sodium lauryl sulfate,sodium salicylate, oleic acid, lecithin, dehydrated alcohol, Tween(e.g., Tween 20, Tween 40, Tween 60, Tween 80 and the like), Span (e.g.,Span 20, Span 40, Span 80 and the like), polyoxyl 40 stearate, polyoxyethylene 50 stearate, edetate disodium, propylene glycol, glycerolmonooleate, fusieates, bile salts, octoxynol and combinations thereof.When used, the concentration of the surfactant in the composition willtypically be from about 0.1% w/w to about 50% w/w. By way of example,concentrations of sodium salicylate, sodium lauryl sulfate and edetatedisodium may be from about 0.01% to about 5% w/w of the composition.Concentrations of polyoxyl 40 stearate, lecithin, dehydrated alcohol,can be from about 0.1% to about 10% w/w of the composition.Concentrations of oleic acid can be from about 0.01% to about 5% w/w ofthe composition. Concentrations of propylene glycol and Tween 20 can befrom about 0.1% to about 25% w/w of the composition.

A pharmaceutically acceptable thickening agent may also be used in thecomposition in order to modify the viscosity of the composition.Numerous pharmaceutically acceptable thickening agents are well-knownand include, for example, methyl cellulose, xanthan gum, carboxymethylcellulose, hydroxypropyl cellulose, carbomer, polyvinyl alcohol,alginates, acacia, chitosans and combinations thereof. The concentrationof the thickening agent will depend upon the agent selected and theviscosity desired. Such agent may be present in the composition at aconcentration of from about 0.1% w/w to about 20% w/w.

A preservative may also be employed to increase the shelf-life of thecomposition. A number of well-known and pharmaceutically acceptablepreservatives may be used in the present composition, including, forexample, parabens, thimerosal, chlorobutanol, benzalkonium chloride, orbenzyl alcohol and combinations thereof. Other ingredients which extendshelf life can be added such as for example, antioxidants. Examples ofantioxidants include sodium metabisulfite, potassium metabisulfite,ascorbyl palmitate and other pharmaceutically acceptable antioxidants.Typically, the antioxidant will be present in the composition in aconcentration of from about 0.01% w/w to about 5% w/w.

Benzyl alcohol and benzalkonium chloride are preferred preservatives. Asuitable concentration of preservative will depend on a number offactors, including, for example, the particular preservative selected,the intended shelf-life of the composition, and the results ofpreservative effectiveness and minimum preservative studies. When used,the concentration of the preservative in the composition will typicallybe from about 0.001% w/w to about 5% w/w, preferably from about 0.01%w/w to about 1% w/w and more preferably from about 0.02% w/w to about0.4% w/w.

In one embodiment of the present invention, the cobalamin compositioncomprises sodium chloride, 0.649% w/w (0.11M); sodium phosphate,monobasic, anhydrous, 0.19% w/w (0.016M); benzyl alcohol, NF, 0.366% w/w(0.034M); sodium hydroxide, NF, 0.04% w/w (0.010M); benzalkoniumchloride, 50%, NF, 0.02% w/w; cyanocobalamin, USP, 0.025% w/w(0.00018M); in purified water, USP, 98.71% w/w.

Alternatively, and in accordance with a preferred embodiment of thepresent invention, the cobalamin composition may be formulated to be asterile, preservative-free composition. While preservatives may extendthe shelf life of a composition, they may also cause or exacerbateirritation to the nasal membranes. Furthermore, because of the frequencywith which the composition of the present invention is preferablyadministered, a bottle of typical volume for storing and dispensing thecomposition will likely be emptied by the patient before the occurrenceof the degradation, spoilage, or bacterial growth that a preservative ismeant to prevent.

Thus in another embodiment, the cobalamin composition ispreservative-free and comprises sodium chloride, 0.649% w/w (0.11M);sodium phosphate, monobasic, anhydrous, 0.19% w/w (0.016M); sodiumhydroxide, NF, 0.04% w/w (0.10M); cyanocobalamin, USP, 0.025% w/w(0.00018M); in purified water, USP, 99.096% w/w.

The composition may be prepared by methods known to those of skill inthe art, including by combining or mixing the components according togenerally accepted procedures. By way of example, the selectedcomponents may be simply mixed in a blender or other standard mixingmachine to produce a concentrated composition which is then adjusted tothe final concentration by the addition of water.

Typically, the cobalamin composition will be stored in and dispensedfrom a sealed container equipped with a metering valve and pump capableof being actuated to deliver or emit an aerosol (e.g., mist or spray) ofthe composition of predetermined volume into the patient's nostril andhaving a suitable droplet size distribution as known to those skilled inthe art. Generally, the size of the droplets are large enough to preventthem from passing directly through the nasal passages and into thelungs, but small enough that they do not coalesce into large drops whicheither run out of the nose or down into the throat.

Suitable containers and metering valves for dispensing the cobalamincomposition according to the methods of the invention are availablecommercially and are known to those of skill in the art. The containerand valve system used to deliver the cobalamin composition mayincorporate any of the conventional aerosol formation techniques. Theseinclude, for example, mechanical pumps; compressed air mechanisms inwhich delivery is made by hand pumping air into the container;compressed gas techniques in which delivery is made by the controlledrelease of a compressed gas (such as, for example, carbon dioxide,nitrogen, and dinitrogen oxide) into the cobalamin containingcomposition; and liquid propellant techniques in which a low boilingliquid hydrocarbon (such as, for example, butane, isobutane, propane,and other low boiling hydrocarbons in either pure or mixed forms),halohydrocarbon, fluorocarbons (such as, for example, FC-152A),chlorofluorocarbons (such as Freon or Freon like fluorocarbons, such as,for example, CFC-11, CFC-12 and CFC-114), and hydrofluorocarbons, alsoreferred to as hydrofluoroalkanes (such as, for example, HFA-134a andHFA-227) are vaporized to exert a pressure and force the compositionthrough the metering valve.

In accordance with a preferred embodiment, and especially when apreservative-free composition is formulated, the cobalamin compositionis stored for administration in a container or bottle including a pumpand metering valve adapted for delivery of a metered spray of thecomposition and designed to inhibit or prevent degradation or spoilageof and bacterial growth in the composition contained therein. Examplesof such a container are the spray pump bottles produced by and availablefrom Pfeiffer (Advanced Preservative Free System) and from Valois (VPY).

The following non-limiting example is provided to further illustrate thepresent invention. It should be appreciated by those skilled in the artthat the techniques disclosed in the example that follows representapproaches the inventors have found function well in the practice of theinvention, and thus can be considered to constitute examples of modesfor its practice. However, those skilled in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments that are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

EXAMPLE

The study comprised twenty-five subjects, ages 18-85, with a history ofdocumented vitamin B₁₂ deficiency who had previously been receivingmaintenance intramuscular (IM) injections of B₁₂. The purpose of thisstudy was to determine whether as an alternative to IM injections, onedaily administration of a 0.025% by weight cobalamin composition issufficient to sustain an efficacious level of B₁₂ in place of IM therapyand, thereby, maintain a serum B₁₂ level within the therapeutic range ofgreater than about 200 ng/L.

As an alternative to an IM injection, an aqueous isotonic compositioncontaining a cobalamin compound was provided to the subjects to allowthem to dose themselves at home with one daily intranasal (IN)administration comprising two puffs or sprays of B₁₂ (cobalamin). Theintranasal cobalamin composition used in this study is listed inTable 1. TABLE 1 Cobalamin Containing Composition Used in Study QuantityRequired per Description % w/w Batch Molarity Sodium Chloride 0.649248.1 gm 0.11 Sodium Phosphate, 0.19 72.6 gm 0.016 Monobasic, anhydrousBenzyl Alcohol, NF 0.366 139.9 gm 0.034 Sodium Hydroxide, NF 0.04 15.3gm 0.010 Benzalkonium 0.02 7.65 gm NA Chloride, 50%, NF (3.83 g BAC +3.83 g (isomeric water) mix) Cyanocobalamin, USP 0.025 9.56 gm 0.00018Purified Water, USP 98.71 37.7 kg NA Total 100 38.2 kg NA

Study Design

This study was an open label, non-randomized, single-arm, activetreatment study. All subjects were instructed to complete daily diariesto record the date and time of administration and any adverse events.

Subjects with chronic B₁₂ deficiencies were treated with intramuscular(IM) injections every 4-8 weeks. At the midpoint of the usual IM therapyinterval (between 2 and 4 weeks post injection) subjects presented atthe clinic for screening and provided blood samples for the measurementof levels of vitamin B₁₂. At the end of the IM therapy interval (4 to 8weeks post injection) the subjects returned to the clinic to provide ablood sample for a low level of B₁₂ measurement and began the intranasal0.025% cyanocobalamin therapy. Each morning during the 8 weekadministration of nasal cyanocobalamin, subjects sprayed 1 spray (0.1mL) of the saline solution containing 0.025% by weight cyanocobalaminfrom an upright metered dose bottle directly into each nostril for a 50μg daily dose of cyanocobalamin.

Subjects returned to the clinic every two weeks (post dosing initiation)for eight weeks to provide blood samples for the evaluation of B₁₂levels.

Primary Efficacy Analysis

The primary efficacy endpoint is the average of the vitamin B₁₂ levelsfrom Visits 3, 4, 5 and 6 relative to the B₁₂ levels at Visit 1. Theratio of each post baseline value to baseline was calculated, and therepeated measures model was used to calculate the means of the ratios ateach visit. The estimate statement (statement 1) provided an estimate ofthe average of the ratios across visits 3, 4, 5 and 6 and the confidenceinterval around that average.

Statistical Analysis

Because the study subjects are on different IM therapy intervals, Visit1 and Visit 2 will not correspond to the same relative week. Thefollowing schedule describes the actual times:

Visit #1 (V1)—Midpoint between IM injections (anywhere from 2 to 4 weekspost IM therapy).

Visit #2 (V2)—Termination of IM therapy (anywhere from 4 to 8 weeks postIM therapy). This is the point at which the next IM injection would havebeen given, but for the fact that the study participants beganintranasal therapy instead.

Visit #3 (V3)—2 weeks post IN therapy initiation.

Visit #4 (V4)—4 week post IN therapy initiation.

Visit #5 (V5)—6 weeks post IN therapy initiation.

Visit #6 (V6)—8 weeks post IN therapy initiation.

Analysis Results

Descriptive Statistics

The average (standard deviation) age of the 25 subjects was 59.8 (15.5)years, ranging from 28.0 to 82.6 years. Seventeen (68.0%) of thesubjects were female. Twenty-one (84.0%) were Caucasian and four (16.0%)were African American. The general pattern was an initial decline fromVisit #1 to Visit #2 and a steady increase in the last four visits(Visits #3-#6), although there were exceptions. The mean B₁₂ value,range, and standard deviation at each visit are summarized in Table 2,and the means and their 95% confidence intervals are also shown inFIG. 1. TABLE 2 SUMMARY OF B₁₂ LEVEL AT EACH VISIT DURING THE STUDY FORTHE 25 SUBJECTS Standard Mean Deviation Range (low-high) Visit N (pg/mL)(pg/mL) (pg/mL) #1 25 484.28 157.93 (419.09, 549.47) #2 25 402.96 149.51(341.24, 464.68) #3 25 505.76 136.73 (449.32, 562.20) #4 25 519.76132.96 (464.88, 574.64) #5 25 510.60 162.41 (443.56, 577.64) #6 25568.28 194.39 (488.04, 648.52)

The above description of the preferred embodiments is intended only toacquaint others skilled in the art with the invention, its principles,and its practical application, so that others skilled in the art mayadapt and apply the invention in its numerous forms, as may be bestsuited to the requirements of a particular use. The present invention,therefore, is not limited to the above embodiments, and may be variouslymodified.

With reference to the use of the word(s) “comprise” or “comprises” or“comprising” in this specification, including the appended claims,unless the context requires otherwise, those words are used on the basisand clear understanding that they are to be interpreted inclusively,rather than exclusively, and that it is intended each of those words tobe so interpreted in construing this specification.

1. A method for maintaining vitamin B₁₂ blood serum levels in a mammal,the method comprising nasally administering to the mammal at least onceevery three days an aqueous composition comprising a cobalamin compoundin an amount sufficient to deliver a dose of no more than about 150 μgof the cobalamin compound to the mammal.
 2. A method for maintainingnormal hematologic status in a pernicious anemia patient followingintramuscular vitamin B₁₂ injection therapy, said method comprisingnasally administering to said patient at least once every three days anaqueous composition comprising a cobalamin compound in an amountsufficient to deliver a dose of no more than about 150 μg of thecobalamin compound to the patient.
 3. The method as set forth in claim 1or claim 2 wherein the aqueous composition comprising a cobalamincompound is administered nasally at least once every two days.
 4. Themethod as set forth in any one of the preceding claims wherein theaqueous composition is isotonic.
 5. The method as set forth in any oneof the preceding claims wherein the aqueous composition comprising acobalamin compound is nasally administered at least once a day.
 6. Themethod as set forth in any one of the preceding claims wherein theaqueous composition comprising a cobalamin compound is nasallyadministered once a day.
 7. The method as set forth in claim 6 whereinthe nasal administration of the aqueous composition comprises a dose ofthe cobalamin compound of no more than about 80 μg.
 8. The method as setforth in claim 6 wherein the nasal administration of the aqueouscomposition comprises a dose of the cobalamin compound of from about 5μg to about 80 μg.
 9. The method as set forth in claim 6 wherein thenasal administration of the aqueous composition comprises a dose of thecobalamin compound of from about 10 μg to about 60 μg.
 10. The method asset forth in claim 6 wherein the nasal administration of the aqueouscomposition comprises a dose of the cobalamin compound of from about 15μg to about 50 μg.
 11. The method as set forth in claim 6 wherein thenasal administration of the aqueous composition comprises a dose of thecobalamin compound of from about 20 μg to about 50 μg.
 12. The method asset forth in claim 6 wherein the nasal administration of the aqueouscomposition comprises a dose of the cobalamin compound of from about 30μg to about 50 μg.
 13. The method as set forth in claim 6 wherein thenasal administration of the aqueous composition comprises a dose of thecobalamin compound of from about 40 μg to about 50 μg.
 14. The method asset forth in claim 6 wherein the nasal administration of the aqueouscomposition comprises a dose of the cobalamin compound of from about 45μg to about 50 μg.
 15. The method as set forth in claim 6 wherein thenasal administration of the aqueous composition comprises a dose of thecobalamin compound of about 50 μg.
 16. The method as set forth in claim6 wherein the nasal administration of the aqueous composition comprisesa dose of the cobalamin compound of no more than about 45 μg.
 17. Themethod as set forth in claim 6 wherein the nasal administration of theaqueous composition comprises a dose of the cobalamin compound of nomore than about 40 μg.
 18. The method as set forth in claim 6 whereinthe nasal administration of the aqueous composition comprises a dose ofthe cobalamin compound of no more than about 35 μg.
 19. The method asset forth in claim 6 wherein the nasal administration of the aqueouscomposition comprises a dose of the cobalamin compound of from about 5μg to about 45 μg.
 20. The method as set forth in claim 6 wherein thenasal administration of the aqueous composition comprises a dose of thecobalamin compound of from about 10 μg to about 45 μg.
 21. The method asset forth in claim 6 wherein the nasal administration of the aqueouscomposition comprises a dose of the cobalamin compound of from about 15μg to about 45 μg.
 22. The method as set forth in claim 6 wherein thenasal administration of the aqueous composition comprises a dose of thecobalamin compound of from about 20 μg to about 45 μg.
 23. The method asset forth in claim 6 wherein the nasal administration of the aqueouscomposition comprises a dose of the cobalamin compound of from about 30μg to about 45 μg.
 24. The method as set forth in claim 6 wherein thenasal administration of the aqueous composition comprises a dose of thecobalamin compound of from about 40 μg to about 45 μg.
 25. The method asset forth in claim 6 wherein the nasal administration of the aqueouscomposition comprises a dose of the cobalamin compound that is about 45μg.
 26. The method as set forth in claim 6 wherein the nasaladministration of the aqueous composition comprises a dose of thecobalamin compound that is about 40 μg.
 27. The method as set forth inclaim 6 wherein the nasal administration of the aqueous compositioncomprises a dose of the cobalamin compound that is about 35 μg.
 28. Themethod as set forth in claim 6 wherein the nasal administration of theaqueous composition comprises a dose of the cobalamin compound that isabout 30 μg.
 29. The method as set forth in claim 6 wherein the nasaladministration of the aqueous composition comprises a dose of thecobalamin compound that is about 25 μg.
 30. The method as set forth inclaim 6 wherein the nasal administration of the aqueous compositioncomprises a dose of the cobalamin compound that is about 20 μg.
 31. Themethod as set forth in claim 6 wherein the nasal administration of theaqueous composition comprises a dose of the cobalamin compound that isabout 15 μg.
 32. The method as set forth in claim 6 wherein the nasaladministration of the aqueous composition comprises a dose of thecobalamin compound that is about 10 μg.
 33. The method as set forth inclaim 6 wherein the nasal administration of the aqueous compositioncomprises a dose of the cobalamin compound that is about 5 μg.
 34. Themethod as set forth in any one of the preceding claims wherein the totalamount of cobalamin compound nasally administered to the mammal is lessthan about 1000 μg per week.
 35. The method as set forth in any one ofthe preceding claims wherein the total amount of cobalamin compoundnasally administered to the mammal is from about 50 μg to about 500 μgper week.
 36. The method as set forth in any one of the preceding claimswherein the total amount of cobalamin compound nasally administered tothe mammal is from about 100 μg to about 450 μg per week.
 37. The methodas set forth in any one of the preceding claims wherein the total amountof cobalamin compound nasally administered to the mammal is from about150 μg to about 400 μg per week.
 38. The method as set forth in any oneof the preceding claims wherein the total amount of cobalamin compoundnasally administered to the mammal is from about 200 μg to about 400 μgper week.
 39. The method as set forth in any one of the preceding claimswherein the total amount of cobalamin compound nasally administered tothe mammal is from about 250 μg to about 350 μg per week.
 40. The methodas set forth in any one of the preceding claims wherein the total amountof cobalamin compound nasally administered to the mammal is from about300 μg to about 350 μg per week.
 41. The method as set forth in any oneof the preceding claims wherein the dose of the cobalamin compound isadministered to the mammal in a plurality of metered sprays of theaqueous composition.
 42. The method as set forth in any one of thepreceding claims wherein the dose of the cobalamin compound isadministered to the mammal in no more than two metered sprays of saidaqueous isotonic composition.
 43. The method as set forth in any one ofthe preceding claims wherein the dose of the cobalamin compound isadministered to the mammal in a single metered spray of the aqueouscomposition.
 44. The method as set forth in any one of the precedingclaims wherein the mammal has an elevated blood metabolite serum levelindicative of a vitamin B₁₂ deficiency.
 45. The method as set forth inany one of the preceding claims wherein the mammal has elevatedhomocysteine, cystathionine, methylmalonic acid or 2-methylcitric acidblood serum levels.
 46. A composition for nasal administration of acobalamin compound, the composition comprising an aqueous solutioncontaining a cobalamin compound and a pharmaceutically acceptablebuffer, the composition having a pH of at least about 6.5.
 47. Thecomposition as set forth in claim 46 wherein the aqueous composition isisotonic.
 48. The composition as set for in claim 46 or 47 wherein thecomposition further comprises a pharmaceutically acceptable additiveselected from the group consisting of tolerance enhancers, absorptionenhancers, preservatives, thickening agents, osmolarity adjusters,complexing agents, stabilizers, solubilizers, and any combinationthereof.
 49. The composition as set forth in claim 46 or 47 wherein thecomposition is free of preservatives.
 50. The composition as set forthin any one of claims 46 to 48 wherein the composition comprises sodiumchloride, 0.649% w/w (0.11M); sodium phosphate, monobasic, anhydrous,0.19% w/w (0.016M); benzyl alcohol, NF, 0.366% w/w (0.034M); sodiumhydroxide, NF, 0.04% w/w (0.010M); benzalkonium chloride, 50%, NF, 0.02%w/w; cyanocobalamin, USP, 0.025% w/w (0.00018M); in purified water, USP,98.71% w/w.
 51. The composition as set forth in claim 49 wherein thecomposition comprises sodium chloride, 0.649% w/w (0.11M); sodiumphosphate, monobasic, anhydrous, 0.19% w/w (0.016M); sodium hydroxide,NF, 0.04% w/w (0.010M); cyanocobalamin, USP, 0.025% w/w (0.00018M); inpurified water, USP, 99.096% w/w.
 52. A composition for nasaladministration of a cobalamin compound, the composition comprising anaqueous solution containing a cobalamin compound and a pharmaceuticallyacceptable buffer and being free of preservatives.
 53. The compositionas set forth in claim 52 wherein the aqueous composition is isotonic.54. The composition as set for in claim 52 or 53 wherein the compositionfurther comprises a pharmaceutically acceptable additive selected fromthe group consisting of tolerance enhancers, absorption enhancers,preservatives, thickening agents, osmolarity adjusters, complexingagents, stabilizers, solubilizers, and any combination thereof.
 55. Thecomposition as set forth in any one of claims 52 to 54 wherein thecomposition comprises sodium chloride, 0.649% w/w (0.11M); sodiumphosphate, monobasic, anhydrous, 0.19% w/w (0.016M); sodium hydroxide,NF, 0.04% w/w (0.010M); cyanocobalamin, USP, 0.025% w/w (0.00018M); inpurified water, USP, 99.096% w/w.
 56. The method or composition as setforth in any one of the preceding claims wherein the cobalamin compoundcomprises cyanocobalamin.
 57. The method or composition as set forth inany one of the preceding claims wherein the cobalamin compound compriseshydroxocobalamin.
 58. The method or composition as set forth in any oneof the preceding claims wherein the composition further comprises apharmaceutically acceptable additive selected from the group consistingof tolerance enhancers, absorption enhancers, preservatives, thickeningagents, osmolarity adjusters, complexing agents, stabilizers,solubilizers, and any combination thereof.
 59. The method or compositionas set forth in any one of the preceding claims wherein the compositionis stored in a container designed to inhibit or prevent degradation of,spoilage of, or bacterial growth in the composition.